The present invention relates to an improved process for stereoselectively synthesizing 2-hydroxyketones using a benzaldehyde lyase.
A thiamine pyrophosphate (TPP)-dependent benzaldehyde lyase, and genetic analyses of the encoding gene, are described in Vicuna et al. (J. Bacteriol., 1989, 171: 2401–2405) and Hinrichsen, P. et al. (Gene, 1994, 144: 137–138). More detailed characterization of the enzyme showed that this benzaldehyde lyase only possesses an irreversible cleaving activity. Thus, for example, two molecules of benzaldehyde are formed when benzoin is used as the starting compound, while anisoin is cleaved into two molecules of anisaldehyde. The possibility of a benzaldehyde lyase catalyzing any linking of C—C compounds is explicitly ruled out.
Decarboxylases or transketolases, which are as a rule likewise dependent on thiamine pyrophosphate (TPP), are particularly suitable for the enzyme-catalyzed synthesis of 2-hydroxyketones.
Whitesides et al. (J. Org. Chem., 1992, 57:5889–5907) and Turner et al. (Tetrahedron Asymmetry, 1996, 7: 2185–2188) describe transketolases which catalyze the cleavage of a 2-hydroxyketone with the simultaneous formation of another 2-hydroxyketone. However, these references do not de-scribe any economic production processes for preparing 2-hydroxyketones using transketolases.
The enzymic synthesis of 2-hydoxyketones in the presence of a TPP-dependent pyruvate decarboxylase has also been described (DE 195 23 269 and DE 297 36 104). However, a disadvantage of the methods which are described is that the pyruvate decarboxylase only accepts a highly restricted substrate spectrum. In addition, the resulting products can spontaneously racemize due to the occurrence of keto-enol tautomerism, resulting in a decrease in the enantioselectivity.
Wilcocks et al. (Biotech. Bioeng., 1992, 39: 1058–1063 and Appl. Env. Microbiol., 1992, 58: 1699–1704) describe using a thiamine pyrophosphate-dependent benzoyl formate decarboxylase to synthesize (S)-2-hydroxy-1-phenylpropanone ((S)-2-HPP) starting from benzoyl formate and acetaldehyde. However, the 2-hydroxyketone which is formed in this case is not enantiomerically pure, either, but is only formed in an enantiomeric excess of 92%. Another disadvantage is that benzyl alcohol is formed as a byproduct when whole cells are used.
The object of the present invention is therefore to make available an improved enzymic process for preparing 2-hydroxyketones, which process no longer suffers from the previously mentioned disadvantages.